Immunoassay method for bnp

ABSTRACT

An immunoassay specific for mammalian γ-BNP derivatives, which uses the first antibody reactive with mammalian α-BNP and the second antibody reactive with mammalian prepro-BNP or γ-BNP derivatives and not α-BNP, and at least one of the first and the second antibodies is optionally labeled detectably labeled or immobilized.

TECHNICAL FIELD

[0001] The present invention relates to an immunoassay for the brainnatriuretic peptide (BNP) which is a member of natriuretic peptidefamily, more specifically, it relates to an immunoassay for γ-BNP andderivatives thereof.

BACKGROUND ART

[0002] Natriuretic peptide family includes three members, i.e., atrialnatriuretic peptide (ANP), brain natriuretic peptide (BNP) and type Cnatriuretic peptide (CNP). Among them, ANP and BNP are cardiac hormoneswhich are mainly biosynthesized in and secreted from the heart. ANP andBNP are similar in structure. ANP is a peptide of 28 amino acids with aring (circular) structure formed by a disulfide bond between the 7th andthe 23rd cysteine residues, while BNP is a peptide of 32 amino acidswith a ring structure formed by a disulfide bond between the 10th andthe 26th cysteine residues. These mature peptides of 28 and 32 aminoacids have been considered to be produced from respective precursor whena leader sequence is cleaved off intracellularly or at the time ofsecretion. That is, there has been reported that human BNP is firstsynthesized as a preprohormone (hereinafter, referred to as prepro-BNP)in myocardial cells, which is split before or at the time of secretionbetween Ser²⁶-His²⁷ to give pro-BNP (hereinafter, referred to as γ-BNP),and which is further split between Arg¹⁰²-Ser¹⁰³ to give BNP-32(hereinafter, referred to as α-BNP) and BNP(1-76), and that the formerexhibits the activity. It has been considered that at least the ringstructure must be remained for the expression of activity.

[0003] The secretion of cardiac hormones being stimulated by variousheart diseases, it well reflects the change in the cardiac functions.The secretion of ANP is accelerated mainly when the atrium undergoes aload, while the biosynthesis and secretion of BNP are stimulated whenthe ventricle undergoes a load. Accordingly, both ANP and BNP are usefulas indicators in the diagnosis of heart disease. As the progress ofinvestigation in the in vivo role of respective hormone, theadvantageous features of BNP as an indicator for diagnosing heartdisease have become clear. For example, the blood concentration of BNPis only ⅙ of ANP in a normal subject but it becomes higher than ANP inpatients of heart failure or the like; the blood concentration of BNPincreases in the case of heart failure like ANP, and the plasmaconcentration of BNP often exceeds that of ANP reflecting moreaccurately the severity of heart dysfunction; the plasma concentrationof both ANP and BNP elevates in peripheral blood and elevation rate ishigher in BNP. Moreover, BNP level in patients of heart failuresometimes increases to several tens times to several hundreds times ofthat of healthy normal subjects, and the change of BNP in the cases ofheart failure is so marked that no other hormones are incomparabletherewith. For these reasons, the usefulness of BNP measurement has beensuggested (Y. Saito et al., Mebio, 12(5), 28, 1995).

[0004] Under the conditions, an immunoassay which utilizes anti-BNPantibody and is applicable to the diagnosis of cardiac insufficiency hasbeen proposed. Japanese Patent Publication (KOHYO) 7-507210 describes amethod of measuring γ-BNP (1-76) produced by biodegradation by proteaseor the like. However, this method is directed to γ-BNP (1-76) whichlacks the portion(s) essential for the expression of activity such asring structure and, therefore, cannot determine the hormone activitydirectly.

[0005] An assay kit for the measurement of α-BNP having natriureticactivity has been marketed (“BNP-32”, Peninsula). With this kit,degradation products of α-BNP in blood including fragments lackingactivity due to the deletion of C-terminal region can also beenmeasured. Taking the low blood concentration of BNP into consideration,the measurements involving the degradation products cannot be disregard.Accordingly, the said method connotes disadvantages to be an assay forBNP in th establishment of an accurate diagnosis of heart failure.

[0006] As a kit for the measurement of BNP free from the disadvantagesabove has been marketed (“SHIONORIA BNP”, Shionogi), whichcharacteristically uses an antibody recognizing the structure essentialfor the expression of activity. However, this method would be affectedsignificantly by the process for collecting and storing blood sample,because α-BNP is extremely instable in collected blood. It is,therefore, suggested that the sample should be specifically treated by,for instance, adding an agent for inhibiting degradation into a bloodcollecting tube or maintaining the sample at low temperature so as toobtain reliable data. Such procedures may hamper the extensive clinicalapplication of the said BNP assay kit.

[0007] DISCLOSURE OF INVENTION

[0008] The present inventors have conducted research intensively for thepurpose of establishing an accurate method of diagnosing cardiacdiseases involving BNP and found that BNP exists in blood in the form ofγ-BNP or its degradation product which at least comprises structurallythe α-BNP moiety (hereinafter, they are referred to as “γ-BNPderivative”), and not in the form of α-BNP which has so far beenconsidered to be dominant. The inventors have also found that γ-BNP ismore stable than α-BNP in blood, that is, one role of the N-terminalstructure of γ-BNP, among many, would be the stabilization of BNP. Theabove indicates that an organism biosynthesizes at least 2 kinds of BNPmolecule which share the BNP activity but differ in half-life. Thesefindings led the present inventors to have a view that it isindispensable to establish a method specific for not only α-BNP but alsoγ-BNP to accomplish an accurate diagnosis of cardiac diseases.

[0009] The present invention provides an immunoassay specific formammalian γ-BNP derivatives, characterized in that it uses the firstantibody which is reactive with mammalian α-BNP and the second antibodywhich is reactive with mammalian prepro-BNP or γ-BNP derivatives and notreactive with α-BNP.

[0010] As used herein, the term “mammalian α-BNP” refers to a peptide oflow molecular weight having natriuretic activity which is derived frommammalian prepro-BNP or γ-BNP through the removal of N-terminal regionas a result of processing at the carboxy terminus of processing signalsequence. In case of human BNP, α-BNP is a peptide consisting ofC-terminal 32 amino acids (Nos. 103-134) of the amino acid sequence ofSEQ ID NO: 1 and having a ring structure. The carboxy terminus ofprocessing signal sequence on the prepro-BNP molecule varies slightlydepending on the species. For example, it is No. 102 Arg in case ofhuman BNP while it is No. 100 amino acid in case of porcine or canineBNP.

[0011] As used herein, the term “mammalian γ-BNP” refers to a pro-BNPcomprising a partial peptide of 32 amino acids corresponding to α-BNP atthe carboxy terminal region. In case of human γ-BNP, it is pro-BNP of108 amino acids from No. 27 His to No. 134 His of the amino acidsequence of SEQ ID NO: 1. The term “prepro-BNP” refers to a peptide of134 amino acids from No. 1 Met to No. 134 His of the amino acid sequenceof SEQ ID NO: 1 in case of human.

[0012] As used herein, the term “mammalian γ-BNP derivative” refers to apeptide fragment derived from mammalian prepro-BNP or γ-BNP throughmainly the in vivo protease reaction, which fragment includes or islarger than α-BNP. Although γ-BNP derivative would comprise a moleculeof the same or smaller size compared to γ-BNP in general, it maycomprise a molecule larger than γ-BNP. Otherwise specifically mentioned,as used herein, the term “γ-BNP derivative” includes γ-BNP itself.

[0013] The term “stable”, when used herein in connection with BNP, meansthat a BNP molecule maintains the C-terminal ring structure includingC-terminus of BNP and the natriuretic activity after undergoing thedegradation by protease, and that the said activity is not significantlydecreased even 24 hours from the collection of blood samples. In lightof this definition, the γ-BNP derivative as the target substance(analyte) of the present immunoassay is stable.

[0014] On the other hand, the term “unstable” means that a BNP sampleundergoes degeneration by protease at the C-terminal region and that thenatriuretic activity is significantly decreases 24 hours from thecollection of blood samples. In light of this definition, α-BNP isunstable.

BRIEF DESCRIPTION OF DRAWINGS

[0015]FIG. 1 is a chromatogram obtained in an α-BNP assay system whereingel filtration HPLC was conducted using Superdex 75 in a plasma sample.In FIG. 1, A indicates the position of elution of α-BNP.

[0016]FIG. 2 is a chromatogram obtained in an α-BNP assay system whereingel filtration HPLC was conducted using Superdex 75 in a plasma sampledifferent from that shown in FIG. 1. In FIG. 2, A indicates the positionof elution of α-BNP.

[0017]FIG. 3 is a chromatogram obtained in an immunoassay specific forγ-BNP wherein gel filtration HPLC was conducted using Superdex 75 in aplasma sample same as those shown in FIG. 2. In FIG. 3, A indicates theposition of elution of α-BNP.

[0018]FIG. 4 is a graph showing the relationships between the storingtime and BNP immunoreactivity of γ-BNP kept in human plasma at 25° C.

[0019]FIG. 5 is a graph showing the relationships between the storingtime and BNP immunoreactivity of α-BNP kept in human plasma at 4° C.

BEST MODE FOR CARRING OUT THE PRESENT INVENTION

[0020] In one embodiment of the present invention, it is related to amethod which uses two antibodies, wherein the first antibody is reactivewith mammalian α-BNP and the second antibody is reactive with prepro-BNPor γ-BNP derivatives and is not reactive with α-BNP.

[0021] Antibodies used in the present method can be monoclonal orpolyclonal antibodies. The first antibody can be prepared according to amethod known in the art using as an antigen human α-BNP which iscommercially available or chemically synthesized, or a partial peptidethereof. Alternatively, a monoclonal antibody appended to a commerciallyavailable α-BNP assay system (kit) for measuring α-BNP (“SHIONORIA”,Shionogi) is also available, which is reactive with the C-terminalregion of α-BNP.

[0022] As the second antibody, any antibody can be used subject that itmeets the conditions above. Preferred examples of such antibody includethose specific for the amino acid sequence shown by the amino acid Nos.27-102 of SEQ ID NO: 1 or metabolites thereof. The γ-BNP derivatives asan analyte to be measured by the present method preferably include atleast the partial amino acid sequence shown by the amino acid Nos.27-134 of SEQ ID NO: 1, in case of human BNP. Accordingly, in apreferred embodiment of the present invention, a special attention ispreferably paid in the selection of an antigen to obtain an antibodycapable of recognizing the amino acid sequence shown by amino acid Nos.27-102. The preparation of such an antibody can be carried out by anyone of methods known in the art. Theoretically, γ-BNP molecule can becleaved by protease at sites corresponding to No. 47 (Arg), No. 53 (Lys)and No. 72 (Arg) in the amino acid sequence of SEQ ID NO: 1, and,therefore, an antibody recognizing an amino acid sequence shown by aminoacid Nos. 73-102 of SEQ ID NO: 1 can be used as the second antibody.

[0023] The assay of the present invention can be either a competitive-or sandwich-assay and an antibody to be used may be a monoclonal- orpolyclonal-antibody.

[0024] At least one of the first and the second antibodies may belabeled detectably or immobilized on a solid support.

[0025] The method for labeling or immobilizing an antibody is known toone ordinary skilled in the art. Examples of label include withoutlimitation radioactive isotopes, enzymes, fluorescent substances,luminescent substances, and particles. The labeling of an antibody canbe carried out according to a method known to one ordinary skilled inthe art, for example, that described by Kono et al. (Kaku-Igaku Gijutu,13(1), 2, (1993)).

[0026] The present invention further provides a kit for immunoassayspecific to mammalian γ-BNP derivatives, characterized in that itcomprises two antibodies wherein the first antibody is reactive withmammalian α-BNP and the second antibody is reactive with mammalianprepro-BNP or γ-BNP derivatives and is not reactive with α-BNP.

[0027] The kit of the present invention can be for a competitive- orsandwich-assay and an antibody to be used may be a monoclonal- orpolyclonal-antibody.

[0028] At least one of the first and the second antibodies may belabeled detectably or immobilized on a solid support. The kit of thepresent invention may further contain a means for detecting the label.Examples of label include without limitation radioactive isotopes,enzymes, fluorescent substances, luminescent substances, or particles.

[0029] The following examples and test examples are provided to furtherillustrate the present invention, without limiting the scope thereof.

EXAMPLE 1

[0030] Measurement of γ-BNP Derivatives by Sandwich IRMA

[0031] Throughout the following Examples, the ordinary reagents used areof special grade supplied by Wako Pure Chemicals Industries, Ltd. orNacalai Tesque, Inc. The bovine serum albumin (BSA) was purchased fromSigma.

[0032] (1) Preparation of Plasma Sample

[0033] 1) Venous blood was collected from patients of cardiac disease orhealthy volunteers and placed in blood-collecting tubes containing EDTAand aprotinin (500 KIU/1, Sigma) derived from bovine lung. The tubeswere centrifuged (×2000 g at 4° C.) for 5 minutes with H-107RGA(Kokusan) to separate blood cells. The resultant plasma samples werefreezed and stored at −80° C. until use.

[0034] 2) The plasma samples prepared in 1) above from patients ofcardiac disease or healthy volunteers were fractionated by gelfiltration HPLC system LC10A (Shimadzu) equipped with Superdex 75 10/30column (Pharmacia). After equilibrating the column with 0.1 M phosphatebuffer (pH 7.5, 0.3M NaCl, 5 mM EDTA) at flow rate of 1 ml/min, 1 ml ofplasma sample was injected and 1 ml each of effluent eluted from thecolumn was collected. Each fraction was subjected to the measurement byassay systems for measuring α-BNP or γ-BNP as described in (2)-2) and(2)-3) below, respectively.

[0035] (2) Construction of Assay System For Measuring α-BNP- or γ-BNPDerivative

[0036] 1) In the assay system, the following peptides, antibodies andkits were used.

[0037] Human α-BNP (Peptide Institute)

[0038] Antibody against the amino terminal region of γ-hBNP (amino acidNos. 27-64 of SEQ ID NO: 1) (Peptide Institute)

[0039] Monoclonal antibody against the carboxy terminal structure ofα-BNP (BC203). BC203 is an immobilized antibody appended to SIONORIA BNPkit (Shionogi), wherein a monoclonal antibody directed to the carboxyterminal structure of α-BNP is immobilized on beads.

[0040] Monoclonal antibody against the ring structure of α-BNP(KYBNPII). KYBNPII is a monoclonal antibody appended to the SIONORIA BNPkit (Shionogi), which is directed to the ring structure (112-128) ofα-BNP, and is labeled with ¹²⁵I.

[0041]2) Measurement of Plasma Fraction by Assay System for α-BNP

[0042] The measurement of α-BNP was carried out by commerciallyavailable “SHIONORIA BNP kit” (Shionogi). The assay is based on sandwichIRMA (Immunoradiometric Assay) which uses a monoclonal antibody KYBNPIIspecific for the ring-structure of α-BNP and another monoclonal antibodyBC203 specific for the carboxy terminal structure of α-BNP. The assaywas carried out in accordance with the supplier's instructions.

[0043] That is, 100 μl each of samples to be assayed or standardsolutions (0, 4, 10, 150, 600 or 2000 pg/ml of α-BNP solution) weredispensed into a polystyrene test tube. To the test tube was added 200μl of iodine-labeled anti-BNP antibody (¹²⁵I) solution, followed by onepolystyrene bead on which anti-BC203 antibody has been immobilized. Themixture was stirred and allowed to react by leaving stand for 18 hoursat 4° C. After washing twice with 2 ml of washing solution,radioactivity was measured on γ-counter ARC-600 (Aloka) The results areshown in FIGS. 1 and 2.

[0044]3) Measurement of Plasma Fraction by Assay System For γ-BNPDerivative

[0045] An antibody against amino terminal portion (Nos. 27-64) of γ-hBNPwas first labeled with ¹²⁵I.

[0046] IgG was purified from anti-serum (Peptide Institute) raisedagainst amino terminal portion (amino acid Nos. 27-64 of SEQ ID NO: 1)of γ-hBNP using MASPII kit (Bio-Rad) and displaced with 0.5 M phosphatebuffer (pH 7.5) using Centricon 30 (Amicon). The labeling of antibodywas carried out by the chloramine T method. To a glass tube wasdispensed 170 μl of purified IgG solution (77.6 μg, IgG), and 10 μl ofNa¹²⁵I solution (34.2 MBq, Amersham) was added. After addition of 0.1%chloramine T solution (20 μl), the mixture was vigorously stirred atroom temperature for 30 seconds. The reaction was quenched by adding 20μl of 0.25% sodium pyrosulfite solution and 20 μl of 5% aqueouspotassium iodide solution. When the reaction mixture was treated withAmpure SA column (Amersham) to remove unreacted ¹²⁵I and to desalt,solution containing ¹²⁵I-labeled antibody was obtained.

[0047] The sandwich IRMA was then carried out in plasma fractions byusing the resultant antibody and polystyrene beads on which an antibodyrecognizing the carboxy terminal structure of α-BNP (BC203).

[0048] To a polystyrene tube was placed 100 μl each of samples to beassayed, followed by 200 μl of 0.1 M phosphate buffer (pH 7.5, 0.3M, 5mM EDTA, 0.2% BSA and 500 KIU/1 bovine lung aprotinin (Sigma)) and onepolystyrene bead on which BC203 antibody has been immobilized. Themixture was stirred and allowed to react by leaving stand for 18 hoursat 4° C. After washing twice with 2 ml of washing solution, 300 μl of¹²⁵I-labeled antibody solution was added. The mixture was stirred andallowed to react by leaving stand for 18 hours at 4° C. After washingtwice with 2 ml of washing solution, radioactivity was measured onγ-counter ARC-600 (Aloka) The results are shown in FIG. 3.

[0049] (3) Results

[0050]FIGS. 1, 2 and 3 show the chromatograms of gel filtration HPLC ofplasma samples obtained from patients, wherein A is the position ofelution of α-BNP.

[0051]FIG. 1 shows the result of the measurement conducted by the α-BNPassay kit described in (2)-2) above. In the FIG. 1, the vertical axisrepresents the concentration of BNP-like substances in each fraction andthe horizontal axis the volume of effluent eluted from the column asmeasured by SHINORIA BNP kit. The solid triangle, open square, and openrhombus respectively represent the measurements in different plasmasamples.

[0052]FIG. 2 shows the result of the measurement conducted by the α-BNPassay kit described in (2)-2) above in samples different from thoseshown in FIG. 1. In the FIG. 2, the vertical axis represents theconcentration of BNP-like substances in each fraction and the horizontalaxis the volume of effluent eluted from the column as measured bySHINORIA BNP kit. The solid triangle and solid square respectivelyrepresent the measurements in different plasma samples.

[0053] From FIGS. 1 and 2, it is revealed that there exist substances ofmolecular weight larger than α-BNP and having BNP-like immunoreactivityin the plasma of patients of cardiac disease, and that they are themajor substances having BNP immunoreactivity.

[0054]FIG. 3 shows the results of the measurement conducted by γ-BNPassay kit described in (2)-3) above in the same samples those shown inFIG. 2. In FIG. 3, the vertical axis represents the radioactivitymeasured by the γ-BNP immunoassay system and the horizontal axis thevolume of effluent eluted from the column. The solid circle representsthe measurements of α-BNP, which obtained after fractionating humanα-BNP solution by HPLC in a similar manner as described in the case ofplasma.

[0055] From FIG. 3, it is revealed that the immunoassay specific forγ-BNP derivative of the present invention can detect the majorsubstances with BNP immunoreactivity, but cannot α-BNP at all.

[0056] The results above indicate that the immunoassay for γ-BNP of thepresent invention is insensitive to α-BNP but specific to γ-BNPderivatives. Further, it has also been revealed that γ-BNP is the majorsubstance having BNP immunoreactivity.

[0057] Test Example 1

[0058] Stability of γ-BNP Derivatives and α-BNP in Plasma

[0059] Fractions suspected to contain γ-BNP derivative were collectedfrom those obtained by treating plasma samples collected from patientsof cardiac disease by gel filtration HPLC. Venous blood was collectedfrom healthy volunteers using blood-collecting tubes containing EDTA inthe absence of bovine lung aprotinin. Plasma samples (the minimumdetection limit of α-BNP<4 pg/ml) were prepared in a manner similar tothat described in (1)-1) above. The plasma sample was allowed to standfor 0, 2, 6, 24 hours at room temperature (25° C.) after addition of thefraction. The stability of BNP derivative was evaluated by determiningthe BNP immunoreactivity in the plasma sample by means of SHIONORIA BNPkit for assaying α-BNP.

[0060] Separately, the stability of α-BNP was evaluated using a plasmasample prepared by adding chemically synthesized α-BNP to plasmacollected from healthy volunteers and standing for 0, 2, 6 and 24 hoursat 4° C. in the absence of bovine lung aprotinin as described above. TheBNP immunoreactivity in the plasma sample was determined by SHIONORIABNP kit in the same manner as above.

[0061] The stability of γ-BNP derivatives and α-BNP in plasma samplesare shown in FIGS. 4 and 5, respectively.

[0062] From FIG. 4, it is revealed that γ-BNP derivatives do not losesignificantly the immunoreactivity compared with the initial activityeven after 24-hour-standing at 25° C. From FIG. 5, by contrast, it isrevealed that α-BNP loses the immunoreactivity to about 40% based on theinitial activity after 24-hour-standing at 4° C.

[0063] The above results demonstrate that α-BNP is far less stablecompared with γ-BNP derivative in blood and that the latter is much moresuited in the diagnosis of cardiac diseases than the former.

INDUSTRIAL APPLICABILITY

[0064] As mentioned above, the BNP level in patients of heart failuresometimes increases to several tens times to several hundreds times ofthat of healthy normal subjects, and the change of BNP in the cases ofheart failure is so marked that no other hormones are incomparabletherewith. For this reason, the usefulness of BNP measurement has beenproposed.

[0065] The immunoassay of the present invention allows to determinespecifically γ-BNP derivatives without measuring α-BNP. Accordingly, thepresent immunoassay can be a clinically significant means for diagnosisand prognostic monitoring of heart failure, which leads toconclusion/judgment somehow different from those resulted fromconventional BNP assay.

[0066] Further, it is herein disclosed for the first time that γ-BNP,which is a target substance to be assayed by the present method, isstable in blood. Therefore, immunoassay of the present inventionprovides stable and reliable clinical data without being affected by theprocess of collecting or storing samples, or the time from thecollection until measurement. Further, the immunoassay of the presentinvention does not require any special pretreatments of blood sample andtherefore gives clinical data conveniently, thereby contributing to theestablishment of highly accurate diagnosis of cardiac diseases.

1 2 1 402 DNA human 1 atg gat ccc cag aca gca cct tcc cgc gcg ctc ctgctc ctg ctc ttc 48 Met Asp Pro Gln Thr Ala Pro Ser Arg Ala Leu Leu LeuLeu Leu Phe 1 5 10 15 ttg cat ctg gct ttc ctg gga ggt cgt tcc cac ccgctg ggc agc ccc 96 Leu His Leu Ala Phe Leu Gly Gly Arg Ser His Pro LeuGly Ser Pro 20 25 30 ggt tca gcc tcg gac ttg gaa acg tcc ggg tta cag gagcag cgc aac 144 Gly Ser Ala Ser Asp Leu Glu Thr Ser Gly Leu Gln Glu GlnArg Asn 35 40 45 cat ttg cag ggc aaa ctg tcg gag ctg cag gtg gag cag acatcc ctg 192 His Leu Gln Gly Lys Leu Ser Glu Leu Gln Val Glu Gln Thr SerLeu 50 55 60 gag ccc ctc cag gag agc ccc cgt ccc aca ggt gtc tgg aag tcccgg 240 Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr Gly Val Trp Lys Ser Arg65 70 75 80 gag gta gcc acc gag ggc atc cgt ggg cac cgc aaa atg gtc ctctac 288 Glu Val Ala Thr Glu Gly Ile Arg Gly His Arg Lys Met Val Leu Tyr85 90 95 acc ctg cgg gca cca cga agc ccc aag atg gtg caa ggg tct ggc tgc336 Thr Leu Arg Ala Pro Arg Ser Pro Lys Met Val Gln Gly Ser Gly Cys 100105 110 ttt ggg agg aag atg gac cgg atc agc tcc tcc agt ggc ctg ggc tgc384 Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys 115120 125 aaa gtg ctg agg cgg cat 402 Lys Val Leu Arg Arg His 130 134 2134 PRT human 2 Met Asp Pro Gln Thr Ala Pro Ser Arg Ala Leu Leu Leu LeuLeu Phe 1 5 10 15 Leu His Leu Ala Phe Leu Gly Gly Arg Ser His Pro LeuGly Ser Pro 20 25 30 Gly Ser Ala Ser Asp Leu Glu Thr Ser Gly Leu Gln GluGln Arg Asn 35 40 45 His Leu Gln Gly Lys Leu Ser Glu Leu Gln Val Glu GlnThr Ser Leu 50 55 60 Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr Gly Val TrpLys Ser Arg 65 70 75 80 Glu Val Ala Thr Glu Gly Ile Arg Gly His Arg LysMet Val Leu Tyr 85 90 95 Thr Leu Arg Ala Pro Arg Ser Pro Lys Met Val GlnGly Ser Gly Cys 100 105 110 Phe Gly Arg Lys Met Asp Arg Ile Ser Ser SerSer Gly Leu Gly Cys 115 120 125 Lys Val Leu Arg Arg His 130 134

1. An immunoassay specific for mammalian γ-BNP derivatives,characterized in that it uses the first antibody reactive with mammalianα-BNP and the second antibody reactive with mammalian prepro-BNP orγ-BNP derivatives and not α-BNP.
 2. The immunoassay of claim 1, whereinthe γ-BNP derivatives comprise the amino acid sequence shown by theamino acid Nos. 27-102 of SEQ ID NO:
 1. 3. The immunoassay of claim 1,wherein the second antibody is specific for the amino acid sequenceshown by the amino acid Nos. 27-102 of SEQ ID NO:
 1. 4. The immunoassayof claim 1 wherein at least one of the first and the second antibodiesis detectably labeled or immobilized.
 5. The immunoassay of claim 1wherein the detectable label is a radioactive isotope, an enzyme, afluorescent substance, a luminescent substance, or a particle.
 6. A kitfor immunoassay specific for mammalian γ-BNP derivatives, characterizein that it comprises the first antibody reactive with mammalian α-BNPand the second antibody reactive with mammalian prepro-BNP or γ-BNPderivatives and not α-BNP.
 7. The kit of claim 6, wherein at least oneof the first and the second antibodies is detectably labeled orimmobilized.
 8. The kit of claim 7 which further comprises a means fordetecting the label.